Prostate Chromatin Research Articles

Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic, and cancerous prostate.

In previous work, we had observed that chromatin-associated nonhistone protein phosphorylation, catalyzed by intrinsic protein kinase reaction in chromatin preparations from human benign prostatic hyperplasia (BPH) prostate samples was markedly elevated, compared with the normal prostate chromatin samples [Rayan et al: Cancer Res 45:2277-2282, 1985]. The properties of this protein kinase reaction were suggestive of the involvement of casein kinase(s). By employing the specific synthetic substrate for casein kinase 2 (CK-2) for assays in cellular fractions, we have shown that this protein kinase is present in human prostate chromatin. Its activity is increased in BPH chromatin by about 25-fold, as compared with its activity in the normal prostate chromatin. This suggests that CK-2 is a possible mediator of the enhanced phosphorylation of chromosomal proteins in BPH chromatin. By comparison, CK-2 activity in chromatin preparations from prostatic carcinoma samples was markedly less elevated than that of the BPH chromatin. Immunohistochemical analysis of the enzyme in human frozen sections of prostate tissue samples showed that the enzyme immunostaining was diffuse in the cytoplasm, but more intense in the nucleus, especially in the nucleoli. In general, the staining corresponded with the enzymic data. However, sections from prostatic carcinoma samples appeared to show differential staining, depending on the Gleason's grade of the sample. The samples with higher Gleason's grade showed less intense immunostain in the nucleus, compared with samples of lower Gleason's grade. Further, regions of sections in samples with higher Gleason's grade did not show any immunostaining. These differences in the characteristics of CK-2 expression in prostatic carcinoma samples may be potentially significant, but need to be evaluated further for their significance to the pathobiology of prostatic neoplasia.

A protein in rat prostatic chromatin interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral prostate chromatin (residual proteins) contain proteins able to interact specifically with androgen-receptor complex and is, therefore, a part of the acceptor complex. Among residual proteins, a 97 KDa protein has been found which binds significantly to a genomic fiagment containing an androgen-regulated gene coding for a 22 KDa protein. The biological significance of this binding in androgen action need to be further studied. A mini-plasmid clone containing 22 KDa protein coding sequence was cloned into Charon 4A genomic library from which a 5.7 Kb genomic fragment was isolated, identified by hybridization with a 5′ and a 3′ cDNA probes, and shown to contain the 5′ flanking sequence. Restriction enzyme treatment of this fragment yielded a 4.7 Kb restriction fragment representing the 5′ upstream region and a 1.0 Kb containing part of the coding sequence. Deletion studies indicated that the 97 KDa protein bound only to a subclone of about 300 bp segment. Furthermore, gel shifting experiment supported its DNA-protein binding.

Activation of a Ca2+-Mg2+-dependent endonuclease as an early event in castration-induced prostatic cell death.

Previous studies have demonstrated that castration-induced androgen withdrawal results in the fragmentation of prostatic DNA into nucleosomal oligomers, and this process comprises an early event in the activation of programed cell death in the rat ventral prostate. This DNA fragmentation could be due to changes in the chromatin conformation increasing its sensitivity to preexisting nucleases and/or to increases in the activity of the nucleases themselves. However, comparative kinetic analysis of in vitro DNA fragmentation induced by exogenous nucleases did not reveal any differences in the sensitivity of prostatic chromatin between intact and castrated rats. In contrast to these negative findings, using [3H] DNA as an exogenous substrate, it was shown that within the first day following castration there was a twofold increase in a Ca2+-Mg2+-dependent nuclease activity without a concomitant increase in other nuclear nucleases. This Ca2+-Mg2+-dependent nuclease activation occurred coincidental with the initial increase in nuclear DNA fragmentation following castration and preceded the enhanced appearance of morphological changes characteristic of dying cells (i.e., apoptosis), as well as the major increase in prostatic DNA loss. These results suggest that castration-induced androgen deprivation leads to a sequential activation of a Ca2+-Mg2+-dependent nuclease leading to the fragmentation of the genome into discrete nucleosomal-sized fragments of DNA, subsequently followed by the fragmentation of the nucleus itself (i.e., apoptosis) and eventually with the complete digestion of the nucleosomal oligomers into component nucleotides (i.e., DNA loss). Since the castration-induced nuclease is dependent upon calcium ions for maximal activity, a potential role of intracellular calcium in the early events activating prostatic cell death was investigated. Acute disturbances in intracellular calcium homeostasis within the ventral prostate by means of a potent calcium influx blocker, nifedipine, simultaneous with castration, resulted in a significant delay in the biochemical and morphological changes associated with prostatic cell death (i.e., prostatic weight loss, prostatic DNA loss, and DNA fragmentation). These results point to a potential role of intracellular calcium levels in the mechanism of activation of castration-induced death of the androgen-dependent epithelial cells in the ventral prostate.

Preferential binding of androgen to transcriptionally active chromatin in rat prostate

Rat ventral prostate chromatin was fractionated by the Micrococcal nuclease procedure of Berkowitz and Riggs ( Biochemistry 20, 7284–7290, 1981). Four chromatin fractions were obtained and analyzed by gel electrophoresis for their protein and DNA components, by hybridization of their DNAs with an androgen regulated cDNA for transcribed DNA sequence, and by [ 3H]dihydrotestosterone exchange assay and steroid competition experiment for specific androgen binding. All four chromatin fractions contained a complete complement of histones and DNA in multiples of about 200 base pairs, indicating repeating nucleosome structure. The active chromatin fraction, which has the shortest nucleosome repeats of 186 basepairs, was enriched in the androgen regulated sequence and bound androgen. These results correlate androgen binding sites with transcribed DNA sequence and structurally distinctive domain of chromatin in the rat prostate.

Correlations between prostate chromatin structure and transcriptional activity and acceptor site distribution.

Androgen-receptor complexes are intimately involved in the maintenance of rat ventral prostate chromatin in a transcriptionally active structure. The presence or absence of androgens influenced the quantity of transcriptionally active chromatin and the distribution of acceptor sites for androgen-receptor complexes as probed by endonucleolytic cleavage. After castration, changes in the sedimentation rates of nucleosome oligomers were consistent with the absence of androgen-receptor complexes and elongating polyribonucleotide chains. These changes were accompanied by decreases in the ability of chromatin released under conditions of minimal nuclease digestion to bind androgen-receptor complexes and to support incorporation of RNA precursors responsive to androgenic stimulation. Saturation analyses of chromatin and nuclear fractions with partially purified androgen-receptor complexes revealed two affinity classes of acceptor sites. After castration, alterations in the intrachromatin distribution of acceptor sites were consistent with their redeployment into areas of decreased nuclease sensitivity, as previously shown for androgen-responsive genes.

Differential localization and androgen sensitivity of prostatic nuclear protein kinases in euchromatin and heterochromatin fractions.

We have examined the distribution and androgenic regulation of protein kinases and phosphoproteins in euchromatin and heterochromatin fractions of rat ventral prostate chromatin. Available procedures to prepare euchromatin and heterochromatin fractions were found to result in the loss of various chromatin-associated protein kinases even though there was no gross change in the gel electrophoretic profile of proteins in these fractions. This loss was prevented by the addition of 0.5 mM phenylmethylsulfonyl fluoride throughout the preparative procedures, which indicates that the protein kinases associated with the chromatin may be particularly susceptible to proteolytic degradation during further subfractionation. By utilizing an improved method for fractionation of chromatin, we have demonstrated a marked enrichment of protein kinase activity (towards phosvitin and endogenous chromosomal proteins) in the euchromatin fraction as compared with heterochromatin. Both of these fractions were also examined for the incorporation of 32P into two main classes of nonhistone proteins (namely, H2SO4-soluble and -insoluble nonhistones). The amount of 32P incorporated into heterochromatin-associated proteins of both classes was markedly less than that in the euchromatin-associated proteins. Protein kinase activities (especially those active towards phosvitin and nonhistone proteins) in the euchromatin fraction as compared with the heterochromatin were significantly reduced within 24 h after androgenic deprivation in the animal. The decreased phosphorylation of nonhistone proteins could be attributed to the loss of endogenous protein kinase activity. The results indicate that not only are chromatin-associated protein phosphokinases preferentially localized in euchromatin fractions but also that these euchromatin-associated protein kinases display the greatest sensitivity to androgenic status of the animal.

In situ cross-linking of androgen receptors to nuclear acceptor sites of rat prostate with formaldehyde.

Androgen receptors were attached covalently in situ to their nuclear acceptor sites with the contact site cross-linker, formaldehyde. Chromatin, prepared from sonicated nuclei of rat prostate, was labeled by isotope exchange with [3H]dihydrotestosterone and found to contain 19,000 +/- 900 (mean +/- S.E.) salt-extractable androgen receptors/nucleus which sedimented in the 3-4 S region of 7.6-76% (v/v) glycerol gradients and at a density of approximately 1.28-1.35 g/ml in CsCl gradients. After incubation of the chromatin with 0.5% (w/v) formaldehyde for 1 h at 4 degrees C, there was a 90% reduction in the concentration of free androgen receptors and an increase in the density of the androgen binding sites recovered from CsCl gradients. Extensive digestion of the cross-linked chromatin with micrococcal nuclease liberated 18% of the androgen receptors as 3-4 S entities and caused an overall decrease in the density of the receptor-acceptor complexes. Ribonuclease digestions had no effect on the androgen receptors cross-linked to chromatin. Mild digestion of the cross-linked preparations with trypsin, alone or in combination with micrococcal nuclease, resulted in the release of 74% and 97% of the androgen receptors, respectively. Together, these findings imply that two classes of receptor-acceptor complexes are present in prostatic chromatin--one, containing about 20% of the androgen receptors in which the receptors are in direct contact with DNA but not with proteins and the other, containing most of the androgen receptors in which the receptors are adjacent to acceptor proteins but not to DNA.

Interaction of androgen receptors with chromatin and DNA

Controlled digestion of rat ventral prostate nuclei by careful adjustment of conditions of temperature, divalent cation concentration, ionic strength and micrococcal nuclease: DNA ratios yielded oligonucleosome fractions corresponding to less than 10% of the total genome which contain the majority of RNA polymerase B activity and androgen-receptor complexes of the nucleus. These parameters were affected acutely by androgen withdrawal and administration: furthermore, such manipulations affected the susceptibility to micrococcal nuclease release of prostate binding protein gene sequences. This transcriptionally-active androgen-influenced fraction was considered ideal for studies of interaction of chromatin components with androgen receptor protein. Androgen receptor was purified approximately 20 000-fold from rat prostate cytosol. The purified protein retained its ability to stimulate RNA polymerase B activity in prostate nuclei and chromatin fractions, and its properties of binding to chromatin and to DNA. However, although purified receptor protein showed tissue-specific binding to prostate chromatin and enhanced binding to fractions released by low nuclease digestion, no such specificity was indicated by binding to total DNA, DNA from specific fractions or cloned prostatic binding protein cDNA.

Androgen regulation of nuclear poly(A) polymerase activities in rat ventral prostate

The existence of nucleoplasmic and chromatin bound forms of poly(A) polymerase in the rat ventral prostate has been demonstrated. The levels of the prostate chromatin and nucleoplasmic poly(A) polymerase activities appeared to be under the influence of testosterone. Castration reduced both free and bound prostatic poly(A) polymerase activities to 30% of the normal values. Administration of testosterone to castrated rat resulted in increases in both nuclear poly(A) polymerase activities at 2–4 h after androgen replcement. The results suggest that post-transcriptional processing is under androgenic control.

Nuclease-sensitivity of prostatic binding protein gene sequences in rat ventral prostate

The ventral prostate nuclei from normal and 3-day castrated rats were subjected to mild micrococcal nuclease digestion. The prostatic binding protein (PBP) gene sequences in the nuclease-sensitive and -resistant DNAs were evaluated by hybridization with DNA probe complementary to PBP-mRNA. The reassociation kinetics showed that the nuclease-sensitive DNA from normal, but not castrated, prostate nuclei was enriched 5.6-fold in the PBP-coding sequences. Digestion of similar nuclei preparations with DNase I to 27% acid-soluble showed no preferential digestion of PBP gene sequences in normal, as compared to castrated, prostate nuclei. Analyses of the levels of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs following castration showed a drastic reduction of nuclear PBP coding sequences at a rate much greater than that of reduction of polysomal PBP-mRNA. These results support that testosterone regulates PBP gene activities at the transcriptional level by altering the conformation of prostate chromatin.

Phosphoproteins and protein kinase activities of ventral prostate chromatin during sexual maturation in the rat

1. 1. In the ventral prostates of rats undergoing normal sexual development, chromatin-associated phosphokinase activities toward dephosphophosvitin and lysine-rich histones were maximal at 48 days of age, whereas phosphorylation of endogenous chromosomal proteins was greatest in the sexually mature adult. 2. 2. Polyacrylamide gel electrophoretic analysis of chromatin proteins from these animals revealed age-dependent changes in the amount of phosphorylation of specific proteins. 3. 3. These were most evident in highly phosphorylated H 2SO 4-soluble nonhistone proteins and in those nonhistones (13–16 K and 20–35 K mol. wt) which are insoluble in H 2SO 4.

Partial purification of nuclear androgen receptor by micrococcal nuclease digestion of chromatin and hydrophobic interaction chromatography.

Extensive (20%) digestion of linker DNA of prostatic chromatin with micrococcal nuclease resulted in the precipitation of 95% of the nuclear androgen receptors. The receptor-enriched precipitate was dissolved in Tes buffer, pH 7.0, containing 0.6--1.2 M NaCl and analysed by hydrophobic interaction chromatography. The adsorption of receptor to omega-amino-alkyl derivatives of agarose increased with the length of the alkyl substituent indicating the presence of hydrophobic regions on the surface of the receptor molecule. Digestion of linker DNA followed by chromatography of precipitated chromatin proteins using 5-aminohexyl-agarose gave rise to a mean 93-fold purificaton of receptor with a recovery of 45%. This approach to the partial separation of nuclear androgen receptor may prove useful in conjunction with more selective purification techniques such as affinity chromatography.

Androgen-induced replication of prostate chromatin DNA: Isolation and electron microscopic visualization of replicative in intermediates

A procedure originally developed to isolate transcriptionally active from less active rat ventral prostate chromatin, employing minimal shear and centrifugation through a dense sucrose gradient, was used to separate prostate chromatin actively synthesizing DNA from total chromatin. It was verified that maximum DNA synthesis in ventral prostates of rats 6 days after castration occurred after administration of testosterone propionate daily for 3 days. When minced ventral prostates from such animals were incubated with [ 3H]thymidine, and the ‘heavy’ and ‘light’ chromatin fractions were separated by sucrose gradient centrifugation, most radioactive DNA was present in the ‘light’ fraction at the top of the gradient. Incorporation was due to DNA synthesis and not to repair, as judged by inhibition with N-ethylmaleimide. Results of in vitro and in vivo thymidine pulse-chase experiments were consistent with initial labelling of DNA-in a replication complex and subsequent sequestration of radioactive DNA in forms resistant to release by the preparative procedure. Although about half the estimtated total endogenous DNA polymerase activity detected in vitro was present in the heavy fraction, the apparent specific activity of the enzyme in the ‘light’ fraction was 5-times as high. Lastly, when equal concentrations of DNA from the separated chromatin fractions were shadowed with platinum-palladium and examined by electron microscopy, 5-times as many Y-shaped structures were seen in the light fraction. This procedure facilitates the isolation of enzymatically active DNA structures undergoing semiconservative replication and study of their subsequent molecular ‘processing’ into forms no longer susceptible to separation by this comparatively gentle method of chromatin preparation. Since the method also yields transcriptionally active chromatin fractions from rat liver and Chinese hamster ovary cell nuclei, it should be applicable to the study of DNA synthesis in these and many other cells.

The loosely bound non-histone chromosomal proteins of rat prostate in androgen action

Rat prostatic chromatin was fractionated by sequential salt extractions into 0.35 M NaCl-soluble (the loosely bound non-histone chromosomal proteins), 2 M NaCl-soluble and -insoluble (the residual proteins) fractions. Homologous and heterologous chromatins were reconstituted with these chromosomal fractions from castrated rats injected with testosterone for 2 h and from castrated control. Chromatin reconstituted with all hormone-treated fractions showed 30% more binding with polylysine and transcriptional template activity than did chromatin reconstituted with castrated fractions. Similar analyses of reconstituted heterologous chromatins indicated that the androgen-induced changes in chromatin were mainly determined by the androgenic state of the 0.35 M NaCl-soluble fraction. The 0.35 M NaCl-soluble fraction also contained 18% more total high mobility group protein than did the corresponding castrated fraction. Limited digestion of 5α-[ 3H]dihydrotestosterone-bound nuclei with pancreatic DNAase 1 to 5% acid-soluble resulted in a rapid release of the nuclear-bound 3H-labelled androgens and protein, in contrast to similar digestion with microsomal nuclease. Since a large portion of the translocated androgen-receptor is bound to protein(s) in the 0.35 M NaCl-soluble fraction, these results suggest that the component(s) in the 0.35 M NaCl-soluble fraction to which androgen-receptor binds is associated with actively transcribing sequences on chromatin.

Isolation of an androgen acceptor from salt extract of rat prostatic chromatin

The soluble androgen acceptor has been isolated from 0.35 M NaCl extract of rat prostatic chromatin by affinity chromatography on DNA-cellulose. The acceptor activity was assayed by interaction with 5α-dihydrotestosterone-receptor. Native DNA enhances this interaction. Polyacrylamide gel electrophoresis of the acceptor under denaturing conditions reveals a single polypeptide of molecular weight of 14,000. Amino acid analysis shows that the acceptor protein contains a higher content of acidic amino acid residues than basic amino acid residues. In an in vitro RNA synthesizing system catalyzed by rat RNA polymerase II, addition of the acceptor stimulates RNA synthesis. Based on incorporation of [γ- 32P]ATP and [γ- 32P]GTP, the stimulation by the acceptor is mainly on the initiation of RNA chains.

Distribution of acceptor sites for androgen-receptor complexes between transcriptionally active and inactive fractions of rat ventral prostate chromatin.

Rat ventral prostate chromatin was separated into two main fractions by controlled digestion with micrococcal nuclease. The soluble fraction obtained after lysis of digested nuclei with EDTA (1 mmol/l), the S2 fraction, represented approximately 17% of the original nuclear DNA, and showed properties consistent with transcriptional activity, i.e. enrichment in nascent RNA, non-histone protein and endogenous RNA polymerase B activity as well as depletion in histones. The fraction sedimented after lysis of nuclei, fraction P, comprised approximately 60% of nuclear DNA, was depleted in nascent RNA, non-histone proteins and endogenous RNA polymerase B activity, but had a higher content of histones. In an attempt to relate the concentration of acceptor sites for androgen-receptor complexes with transcriptional activity, it was shown that the S2 fraction was enriched in these acceptor sites. However, if measurements were based on the intact cell the transcriptionally inactive portion contained 2.5--3 times as many 'acceptor' sites, although these sites had lower affinity for androgen-receptor complexes than had those in the transcriptionally active fraction.

Regulation of transcription of the prostate genome by androgens

The effect of androgen withdrawal on a number of parameters of transcription has been studied using reconstituted systems based on rat ventral prostate chromatin or nuclei. Nuclear receptors, total RNA polymerase activity, the numbers of RNA polymerase molecules active in transcription, RNA-chain lengths and the rates of their elongation are all significantly decreased 1 day after castration and continue to decrease, less dramatically, until 7 days after castration. Both α-amanitin-sensitive and -insensitive RNA polymerase activities were affected. The changes in initiation sites on chromatin available to RNA polymerase B do not correlate with alterations in receptor levels and RNA synthesis in intact nuclei. Possible reasons for these discrepancies are discussed. Hybridization of cellular poly(A)-containing RNA from prostates of normal rats and rats castrated 3 days previously suggested that castration resulted in the reduced concentration of a small number of highly-abundant poly(A) RNA sequences.

Binding of androgen receptor to prostatic chromatin requires intact linker DNA.

Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.

Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves.

The role of nonhistone chromosomal proteins in the interaction of prostate chromatin with androgen · receptor complex

Rat prostate nonhistone chromatin proteins were fractionated by sequential extraction with 0.35 M NaCl and 2.0 M NaCl into 0.35 M NaCl-soluble, 2 M NaCl-soluble and -insoluble (residual) fractions. Interaction of [ 3H]dihydrotestosterone · receptor complex with reconstituted chromatin lacking one of these fractions and with reconstituted hybrid chromatin using spleen fractions indicate that binding of chromatin to androgen · receptor is largely determined by the residual fraction. The residual fraction of prostate also interacts directly with [ 3H]dihydrotestosterone · receptor complex target tissue-specifically similar to prostate chromatin. The DNA in the residual fraction is mostly bound by nonhistone proteins and the template activity of the residual DNA in transcription is severely restricted. These characteristics are discussed in relation to the acceptor role of the residual fraction in androgen action.